Use of predetermined nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules

ABSTRACT

The present invention provides improved methods for amplifying a nucleic acid molecule. More specifically, the invention provides methods for nucleic acid amplification which use primers having equivalent priming efficiency.

FIELD OF THE INVENTION

[0001] The present invention is in the field of recombinant DNAtechnology. This invention is directed to a process for amplifying anucleic acid molecule, and to the molecules employed and producedthrough this process.

BACKGROUND OF THE INVENTION

[0002] Assays capable of detecting the presence of a particular nucleicacid molecule in a sample are of substantial importance in forensics,medicine, epidemiology and public health, and in the prediction anddiagnosis of disease. Such assays can be used, for example, to identifythe causal agent of an infectious disease, to predict the likelihoodthat an individual will suffer from a genetic disease, to determine thepurity of drinking water or milk, or to identify tissue samples. Thedesire to increase the utility and applicability of such assays is oftenfrustrated by assay sensitivity. Hence, it would be highly desirable todevelop more sensitive detection assays.

[0003] Nucleic acid detection assays can be predicated on anycharacteristic of the nucleic acid molecule, such as its size, sequence,and, if DNA, susceptibility to digestion by restriction endonucleases,etc. The sensitivity of such assays may be increased by altering themanner in which detection is reported or signaled to the observer. Thus,for example, assay sensitivity can be increased through the use ofdetectably labeled reagents. A wide variety of such labels have beenused for this purpose. Kourilsky et al. (U.S. Pat. No. 4,581,333)describe the use of enzyme labels to increase sensitivity in a detectionassay. Radioisotopic labels are disclosed by Falkow et al. (U.S. Pat.No. 4,358,535), and by Berninger (U.S. Pat. No. 4,446,237). Fluorescentlabels (Albarella et al., EP 144914), chemical labels (Sheldon III etal., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.4,563,417), modified bases (Miyoshi et al., EP 119448), etc. have alsobeen used in an effort to improve the efficiency with which detectioncan be observed.

[0004] Although the use of highly detectable labeled reagents canimprove the sensitivity of nucleic acid detection assays, thesensitivity of such assays remains limited by practical problems whichare largely related to nonspecific reactions which increase thebackground signal produced in the absence of the nucleic acid the assayis designed to detect. Thus, for some applications, such as for theidentification of a pure culture of a bacteria, etc., the concentrationof the desired molecule will typically be amenable to detection,whereas, for other potential applications, the anticipated concentrationof the desired nucleic acid molecule will be too low to permit itsdetection by any of the above-described assays.

[0005] In response to these impediments, a variety of, highly sensitivemethods for DNA amplification have been developed.

[0006] One, method for overcoming the sensitivity limitation of nucleicacid concentration is to selectively amplify the nucleic acid moleculewhose detection is desired prior to performing the assay. RecombinantDNA methodologies capable of amplifying purified nucleic acid fragmentshave long been recognized. Typically, such methodologies involve theintroduction of the nucleic acid fragment into a DNA or RNA vector, theclonal amplification of the vector, and the recovery of the amplifiednucleic acid fragment; Examples of such methodologies are provided byCohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, etc.

[0007] Other known nucleic acid amplification procedures includetranscription based amplification systems (Kwoh, D. et al., Proc. Natl.Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras T R et al., PCT appl. WO88/10315 (priority: U.S. patent applications Ser. Nos. 064,141 and202,978)). Schemes based on ligation of two (or more) oligonucleotidesin the presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu, D. Y. et al., Genomics 4:560 (1989)).

[0008] Miller, H. I. et al., PCT appl. WO 89/06700 (priority: U.S.patent application Ser. No. 146,462, filed Jan. 21, 1988), disclose anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme was not cyclic; i.e. new templates were not produced from theresultant RNA transcripts.

[0009] Davey, C. et al. (European Patent Application Publication No.329,822) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA). The ssRNA is a first template for a firstprimer oligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNaseH, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′-to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0010] Methods that include a transcription step, e.g. that of Davey, C.et al. (European Patent Application Publication No. 329,822), canincrease product by more than a factor of 2 at each cycle. Indeed, as100 or more transcripts can be made from a single template, factors ofincrease of 100 or more are theoretically readily attainable.Furthermore, if all steps are performed under identical conditions, nomolecule which has finished a particular step need “wait” beforeproceeding to the next step. Thus amplifications that are based ontranscription and that do not require thermocycling are potentially muchfaster than thermo cycling amplifications which are based ontemplate-dependent primer extension.

[0011] In methods which amplify a nucleic acid molecule by templatedependent extension, the nucleic acid molecule is used as a template forextension of a nucleic acid primer in a reaction catalyzed bypolymerase. For example, Panet and Khorana (J. Biol. Chem. 249:5213-5221(1914) which reference is incorporated herein by reference) demonstratedthe replication of deoxyribopoly-nucleotide templates bound tocellulose. Kleppe et al. (J. Mol. Biol. 56:341-361 (1971) whichreference is incorporated herein by reference) disclosed the use ofdouble- and single-stranded DNA molecules as templates for the synthesisof complementary DNA.

[0012] The most widely used method of nucleic acid amplification, the“polymerase chain reaction” (“PCR”), involves template dependentextension (Mullis; K. et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017,EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No.4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al.,U.S. Pat. No. 4,683,194), which references are incorporated herein byreference). PCR achieves the amplification of a specific nucleic acidsequence using two oligonucleotide primers complementary to regions ofthe sequence to be amplified. Extension products incorporating theprimers then become templates for subsequent replication steps.

[0013] The polymerase chain reaction provides a method for selectivelyincreasing the concentration of a nucleic acid molecule having aparticular sequence even when that molecule has not been previouslypurified and is present only in a single copy in a particular sample.The method can be used to amplify either single or double stranded DNA.The essence of the method involves the use of two oligonucleotides toserve as primers for the template-dependent, polymerase mediatedreplication of the desired nucleic acid molecule.

[0014] The precise nature of the two oligonucleotide primers of the PCRmethod is critical to the success of the method. As is well known, amolecule of DNA or RNA possesses directionality, which is conferredthrough the 5′→3′ linkage of the sugar-phosphate backbone of themolecule; Two DNA or RNA molecules maybe linked together through theformation of a phosphodiester bond between the terminal phosphate groupof one molecule and the terminal 3′ hydroxyl group of the secondmolecule. Polymerase dependent amplification of a nucleic acid moleculeproceeds by the addition of a 5′ nucleoside triphosphate to the 3′hydroxyl end of a nucleic acid molecule. Thus, the action of apolymerase extends the 3′ end of a nucleic acid molecule. These inherentproperties are exploited in the selection of the two oligonucleotideprimers of the PCR. The oligonucleotide sequences of the two primers ofthe PCR method are selected such that they contain sequences identicalto, or complementary to, sequences which flank the sequence of theparticular nucleic acid molecule whose amplification is desired. Morespecifically, the nucleotide sequence of the “first” primer is selectedsuch that it is capable of hybridizing to an oligonucleotide sequencelocated 3′ to the sequence of the desired nucleic acid molecule, whereasthe nucleotide sequence of the “second” primer is selected such that itcontains a nucleotide sequence identical to one present 5′ to thesequence of the desired nucleic acid molecule. Both primers possess the3′ hydroxyl groups which are necessary for enzyme mediated nucleic acidsynthesis.

[0015] In the polymerase chain reaction, the reaction conditions arecycled between those conducive to hybridization and nucleic acidpolymerization, and those which result in the denaturation of duplexmolecules. In the first step of the reaction, the nucleic acids of thesample are transiently heated, and then cooled, in order to donaitureany double stranded molecules which may be present. The “first” and“second” primers are then added to the sample at a concentration whichgreatly exceeds that of the desired nucleic acid molecule. When thesample is incubated under conditions conducive to hybridization andpolymerization, the “first” primer will hybridize to the nucleic acidmolecule of the sample at a position 3′ to the sequence of the desiredmolecule to be amplified. If the nucleic acid molecule of the sample wasinitially double stranded, the “second” primer will hybridize to thecomplementary strand of the nucleic acid molecule, at a position 3′ tothe sequence of the desired molecule which is the complement of thesequence whose amplification is desired. Upon addition of a polymerase,the 3′ ends of the “first” and (if the nucleic acid molecule was doublestranded) “second” primers will be extended. The extension of the“first” primer will result in the synthesis of a DNA molecule having theexact sequence of the complement of the desired nucleic acid. Extensionof the “second” primer will result in the synthesis of a DNA moleculehaving the exact sequence of the desired nucleic acid.

[0016] The PCR reaction is capable of exponential amplification ofspecific nucleic acid sequences because the extension product of the“first” primer contains a sequence which is complementary to a sequenceof the “second” primer, and thus will serve as a template for theproduction of an extension product of the “second” primer. Similarly,the extension product of the “second” primer, of necessity, contain asequence which is complementary to a sequence of the “first” primer, andthus will serve as a template for the production of an extension productof the “first” primer. Thus, by permitting cycles of hybridization,polymerization, and denaturation, a geometric increase in theconcentration of the desired nucleic acid molecule can be achieved.Reviews of the polymerase chain reaction are provided by Mullis, K. B.(Cold Spring Harbor Symo. Quant. Biol. 51:263-273 (1986)); Saiki, R. K.,et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al.(Met. Enzymol. 155:335-350 (1987), which references are incorporatedherein by reference).

[0017] PCR technology is useful in that it can achieve the rapid andextensive amplification of a polynucleotide molecule. However, themethod requires the preparation of two different primers which hybridizeto two oligonucleotide sequences flanking the target sequence. Theconcentration of the two primers can be rate limiting for the reaction.Although it is not essential that the concentration of the two primersbe identical, a disparity between the concentrations of the two primerscan greatly reduce the overall yield of the reaction.

[0018] All of the above amplification procedures depend on the principlethat an end product of a cycle is functionally identical to a startingmaterial. Thus, by repeating cycles, the nucleic acid is amplifiedexponentially.

[0019] Methods that use thermocycling, e.g. PCR or Wu, D. Y. et al.(Genomics 4:560 (1989)), have a theoretical maximum increase of productof 2-fold per cycle, because in each cycle a single product is made fromeach template. In practice, the increase is always lower than 2-fold.Further slowing the amplification is the time spent in changing thetemperature. Also adding delay is the need to allow enough time in acycle for all molecules to have finished a step. Molecules that finish astep quickly must “wait” for their slower counterparts to finish beforeproceeding to the next step in the cycle; to shorten the cycle timewould lead to skipping of one cycle by the “slower” molecules, leadingto a lower exponent of amplification.

[0020] One disadvantage of PCR is that it requires the use of twoprimers, and thus requires that sequence information be available fortwo regions of the target molecule. This is often a significantconstraint. In some situations, only the amino acid sequence encoded bya target sequence is known. To amplify the target sequence, it isnecessary to employ sets of degenerate primers (corresponding to each ofthe possible sequences capable of encoding the amino acid sequence codedfor by the two regions of the target molecule). The use of suchdegenerate primer sets can cause significant priming errors, and thus andecrease amplification efficiency. One means of decreasing the number ofmembers in the primer sets when conducting PCR Amplification is throughthe use of primers containing deoxyinosine at positions of ambiguity(Patil, R. V., Nucl. Acids Res. 18:3080 (1990); Fordham-Skelton, A. P.,et al., Molec. Gen. Genet. 221:134-138 (1990); both of which referencesare herein incorporated by reference).

[0021] A second significant disadvantage of the PCR reaction is thatwhen two different primers are used, the reaction conditions chosen mustbe selected such that both primers “prime” with similar efficiency.Since the two primers necessarily have different sequences, thisrequirement can constrain the choice of primers and require considerableexperimentation. Furthermore, if one tries to amplify two differentsequences simultaneously using PCR (i.e. using two sets of two primers),the reaction conditions must be optimized for four different primers.

SUMMARY OF THE INVENTION

[0022] The present invention provides an improved method for equalizingthe hybridization efficiency of the primers used in a PCR reaction. Itthus comprises an improvement in PCR amplification. The inventionachieves this goal by employing a primer molecule which containspre-determined nucleotides having altered base pairing characteristics.

[0023] In detail, the invention provides a method for amplifying theconcentration of a nucleic acid molecule using two primers, comprisingthe steps:

[0024] (a) performing the template-dependent extension of a firstprimer, the primer being hybridized to a first strand of the molecule,wherein the extension forms a second strand of a nucleic acid moleculecomplementary to the first strand;

[0025] (b) performing the template-dependent extension of the secondstrand, by extending a second primer, the primer being hybridized to thesecond strand of the molecule, wherein the extension forms a copy of thefirst strand of the nucleic acid molecule;

[0026] (c) performing the template-dependent extension of the copy ofthe first strand, to thereby form a copy of the second strand of thenucleic acid molecule;

[0027] (d) repeating steps (a), (b), and (c), to thereby achieve theamplification of the nucleic acid molecule; wherein at least one of thefirst and second primers contains at least one deoxyinosine residue, andwherein the first and second primers have equivalent efficiency ofprimer extension.

[0028] The invention also provides the embodiments of the above methodwherein the nucleic acid molecule is an RNA or a DNA molecule, andwherein such molecule is either single-stranded or double-stranded.

[0029] The invention also provides the embodiments of the above methodswherein only one of the primers contains at least one deoxyinosineresidue, and wherein both of the primers contain at least onedeoxyinosine residue.

[0030] The invention also provides the embodiment of the above methodswherein the nucleic acid molecule being amplified is polyadenylated atits 3′ end, and wherein one of the primers contains a poly-T sequence,and the other of the primers contains at least one deoxyinosine residue.

[0031] The invention also provides the embodiment of the above methodswherein the nucleic acid molecule being amplified, copy thereof orcomplementary copy thereof has been extended to contain a 3′ sequence,and wherein one of the primers is capable of hybridizing to the 3′sequence, the primer containing at least one, deoxyinosine residue.

[0032] The invention also provides the embodiment of the above methodswherein at least one of the primers is extended using a thermostable DNApolymerase, such as Taq polymerase.

[0033] The invention also provides a kit for amplifying a nucleic acidmolecule-containing:

[0034] a first container containing a primer, the primer being capableof hybridizing to the nucleic acid molecule, and containing at least onedeoxyadenosine residue; and

[0035] a second container containing an enzyme capable of adding a Cnucleotide to the nucleic acid molecule, the C nucleotide being capableof base pairing with the deoxyinosine residue of the primers.

[0036] The invention also provides for the above kit which additionallycontains a third container containing a thermostable DNA polymerase,such as Taq polymerase capable of extending the primer of the firstcontainer, when the primer is hybridized to a sequence containing the Cresidue added by the enzyme of the second container.

[0037] The invention also provides a kit for amplifying a nucleic acidmolecule containing:

[0038] a first container containing a first primer, the primer beingcapable of hybridizing to the nucleic acid molecule, and containing atleast one deoxyinosine residue; and

[0039] a second container containing a second primer; whereintemplate-dependent extension of the first primer produces a secondnucleic acid molecule which is capable of hybridizing to the secondprimer, and wherein template-dependent extension of the second primerproduces a copy of the first nucleic acid molecule. The invention alsoprovides the above kit which additionally contains a third containercontaining a thermostable DNA polymerase, such as Taq polymerase,capable of extending either the primer of the first container, or theprimer of the second container when the primer is hybridized to anucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

[0040]FIG. 1 shows a depiction of the 3′ RACE reaction.

[0041]FIG. 2 shows a depiction of the 5′ RACE reaction.

[0042]FIG. 3 shows the use of inosine in the 3′ RACE reaction.

[0043]FIG. 4 shows the use of inosine in the 5′ RACE reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention provides an improved method for amplifyinga desired nucleic acid molecule in a sample. Such samples may includebiological samples derived from a human or other animal source (such as,for example, blood, stool, sputum, mucus, serum, urine, saliva,teardrop, a biopsy sample, an histology tissue sample, a PAP smear, amole, a wart, an agricultural product, waste water, drinking water,milk, processed foodstuff, air, etc.) including samples derived from abacterial or viral preparation, as well as other samples (such as, forexample, agricultural products, waste or drinking water, milk or otherprocessed foodstuff, air, etc.).

[0045] The method, provided by the present invention, for amplifying adesired nucleic acid molecule in a sample, may be used to amplify anydesired nucleic acid molecule. Such molecules may be either DNA or RNA.The molecule may be in either a double-stranded or single-stranded form.However, if the nucleic acid is double-stranded at the start of theamplification reaction it is preferably first treated to render the twostrands into a single-stranded, or partially single-stranded, form.Methods are known to render double-stranded nucleic acids intosingle-stranded, or partially single-stranded, forms,. such as heating,or by alkali treatment, or by enzymatic methods (such a by helicaseaction, etc.), or by binding proteins, etc. General methods foraccomplishing this treatment are provided by Maniatis, T., et al. (In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1982)), and by Haymes, B. D., et al. (In:Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985)), which references are herein incorporated by reference.

[0046] Macromolecular entities that contain nucleic acid other thandouble-stranded DNA, or single-stranded DNA, such as single-strandedRNA, double-stranded RNA or mRNA are capable of being amplified by themethod of the invention. For example, the RNA genomes of certain virusescan be converted to DNA by reaction with enzymes such as reversetranscriptase (Maniatis, T. et al., Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory, 1982; Noonan, K. F. et al.,Nucleic Acids Res. 16:10366 (1988)). The product of the reversetranscriptase reaction may then be amplified according to the invention.

[0047] The nucleic acid molecules which may be amplified in accordancewith the present invention may be homologous to other nucleic acidmolecules present in the sample (for example, it may be a fragment of ahuman chromosome isolated from a human cell biopsy, etc.).Alternatively, the molecule may be heterologous to other nucleic acidmolecules present in the sample (for example, it may be a viral,bacterial, or fungal nucleic acid molecule isolated from a sample ofhuman blood, stools, etc.). The methods of the invention are capable ofsimultaneously amplifying both heterologous and homologous molecules.For example, amplification of a human tissue sample infected with avirus may result in amplification of both viral and human sequences.

[0048] The present methods do not require that the molecules to beamplified have any particular sequence or length. In particular, themolecules which may be amplified include any naturally occurringprocaryotic (for example, pathogenic or non-pathogenic bacteria,Escherichia, Salmonella, Clostridium, Agrobacter, Staphylococcus andStreptomyces, Streptococcus, Rickettsiae, Chlamydia, Mvcotlasma, etc.),eukaryotic (for example, protozoans and parasites, fungi, yeast, higherplants, lower and higher animals, including mammals and humans) or viral(for example, Herpes viruses, HIV, influenza virus, Epstein-Barr virus,hepatitis virus, polio virus, etc.) or viroid nucleic acid. The nucleicacid molecule can also be any nucleic acid molecule which has been orcan be chemically synthesized. Thus, the nucleic acid sequence may ormay not be found in nature.

[0049] “Primer” as used herein refers to a single-strandedoligonucleotide or a single-stranded polynucleotide that is extended bycovalent addition of nucleotide monomers during amplification. Nucleicacid amplification often is based on nucleic acid synthesis by a nucleicacid polymerase. Many such polymerases require the presence of a primerthat can be extended to initiate such nucleic acid synthesis. A primeris typically 11 bases or longer; most preferably, a primer is 17 basesor longer.

[0050] “Reaction” denotes a liquid suitable for conducting a desiredreaction (such as amplification, hybridization, cDNA synthesis, etc.).

[0051] “Amplification” as used herein refers to an increase in theamount of the desired nucleic acid molecule present in a sample.“Substantial amplification” refers to greater than about threefoldamplification. Any of the primer extension amplification methodsdiscussed above may be improved in accordance with the presentinvention.

[0052] As used herein, two sequences are said to be able to hybridize oranneal to one another if they are capable of forming an anti-paralleldouble-stranded nucleic acid structure. Conditions of nucleic acidhybridization suitable for forming such double stranded structures aredescribed by Maniatis, T., et al. (In: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982)), and by Haymes, B. D., et al. (In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985), both hereinincorporated by reference). Two sequence are said to be “complementary”to one another if they are capable of hybridizing to one another to forma stable anti-parallel double-stranded nucleic acid structure. Thus, thesequences need not exhibit precise complementarity, but need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure. Thus, departures from completecomplementarity are permissible, so long as such departures are notsufficient to completely preclude hybridization to form adouble-stranded structure. Hybridization of a primer to a complementarystrand of nucleic acid is a prerequisite for its template-dependentpolymerization with polymerases. Factors (see Maniatis, T., et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1982), and Haymes, B. D., et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985))which affect the base pairing of primers to their complementary nucleicacids subsequently affect priming efficiency (i.e. the relative rate ofthe initiation of priming by the primer). The nucleotide composition ofa primer can affect the temperature at which annealing is optimal andtherefore can affect its priming efficiency.

[0053] The methods of the present invention permit one to adjust thehybridization efficiency of the primers used to amplify a nucleic acidmolecule. Such adjustment may either increase or decrease the differencebetween the respective hybridization efficiencies of two primers. Themethods of the present invention thus permit one to equalize therespective hybridization efficiencies of the two primers. Severalfactors must be considered in order to determine the efficiency ofhybridization between a primer and a target molecule. At the simplestlevel, the efficiency is determined by the length of the primer, theconcentration of the primer, the temperature and the ionic strength ofthe reaction. It is also influenced by the sequence complexity of theprimer, and specifically, by the number of hydrogen bonds which willform between the primer and the template. As stated above, the basepairing of an A to a T will form two hydrogen bonds; the base pairing ofa G to a C will form three hydrogen bonds. Thus, at a firstapproximation, it is possible to more closely hybridize two differentprimers to two regions of a target molecule by adjusting the length andsequence complexity of the primers so that they contain the same numberof hydrogen bonds. Unfortunately, additional factors, such as secondarystructure, stacking energy, cooperativity in binding, etc, complicatethe analysis. Thus, a determination of the conditions needed to ensurethat two primers hybridize with equal efficiency requires a multifactoranalysis. Methods for determining relative primer efficiency aredisclosed by Breslauer, K. J. et al. (Proc. Natl. Acad. Sci. (U.S.A.)83:3746-3750 (1986)), Freier, S. M. et al. (Proc. Natl. Acad. Sci.(U.S.A.) 83:9373-9377 (1986)), Rychlik, W. et al. (Nucl. Acids Res.17:8543-8551 (1989)), Lathe, R. (J. Molec. Biol. 183:112 (1986)),Sambrook, J. et al. (Molecular Cloning (A Laboratory Manual), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), vol. 2, pp.1118), Schildkraut, C. et al. (Biopolymers 3:195-208 (1965)), Baldino,F. et al. (Meth. Enzymol. 168:761-777 (1989)), and in the Handbook ofBiochemistry and Molecular Biology (Fasman, G. D., Ed.), Third Edition(1975), Nucleic Acids, Vol. 1, pp. 589, CRC Press, Cleveland, Ohio; allof which references are herein incorporated by reference. Mostpreferably, a determination of relative primer efficiency is performedusing a computer program, such as “Oligo™ Primer Analysis Software(National Biosciences, Inc., Hamel, Minn.).

[0054] The improvement provided by the present invention results fromusing “pre-determined” nucleotides having altered base pairingcharacteristics in at least one of the primer molecules to equalize theefficiency of hybridization between (1) that primer molecule and itscomplement sequence on the target molecule, and (2) a second primermolecule and its complement sequence on the target molecule. The primersof the present invention are preferably 15-50 residues in length,although shorter or longer primer sequences can be employed.

[0055] DNA typically contains a polynucleotide composed of the 4“natural” bases: A (adenine), T (thymine), C (cytosine), and G(guanine). The hydrogen bonding (or base pairing) among thesenucleotides creates the double-stranded structure of a DNA molecule. AnA-containing residue base pairs to a T-containing residue through theformation of two hydrogen bonds; a G-containing residue base pairs to aC-containing residue through the formation of three hydrogen bonds.

[0056] The term “pre-determined nucleotides having altered base pairingcharacteristics” is intended to refer to nucleotides which have basesother than the A, T, C or G naturally found in DNA. Although thepre-determined nucleotides will be capable of hydrogen bonding withnaturally occurring nucleotides (such as the A, T, C or G-containingnucleotides of the template), it will form fewer hydrogen bonds withsuch nucleotides than would other naturally occurring nucleotides.

[0057] A nucleotide containing deoxyinosine (“dI”) is a preferredexample of a such a pre-determined nucleotide containing the base,inosine. It is capable of forming two hydrogen bonds with either A, C,T, or G. (Barker, R., Orcanic Chemistry of Biolocical Molecules,Prentice-Hall, N.J. (1971)). Thus, in a preferred embodiment, when I isused In a primer (or template) in lieu of G or, in lieu of C, the basepairing efficiency is altered.

[0058] Other examples of “pre-determined” nucleotides are those whichcontain hypoxanthine or xanthine (each useful, for example, in lieu ofG, to form two hydrogen bonds when base pairing with C), or those whichcontain methylated derivatives of naturally occurring bases (forexample, 7-methylguanine, etc.).

[0059] In accordance with the methods of the present invention, nucleicacid amplification, such as through two primer mediated PCR, is achievedusing at least one primer containing at least one of the above-described“pre-determined” nucleotides. The position, type and number of“pre-determined” nucleotide(s) in the primer sequence containing the“pre-determined” nucleotide are selected such that the efficiency ofprimer extension of that primer is equivalent to the efficiency ofprimer extension of the second primer.

[0060] As used herein, the term “equivalent efficiency of primerextension” is intended to refer to the ability of one primer, relativeto a second primer, to hybridize to a complementary sequence on atemplate molecule, and to serve as a substrate for template-dependentprimer extension by a DNA or RNA polymerase. Primer extension is said tobe “template dependent” when the sequence of the newly synthesizedstrand of nucleic acid is dictated by complementary base pairing. A“polymerase” is an enzyme that is capable of incorporating nucleosidetriphosphates to extend a 3′ hydroxyl group of a nucleic acid molecule,if that molecule has hybridized to a suitable template nucleic acidmolecule. Polymerase enzymes are discussed in Watson, J. D., In:Molecular Bioloy of the Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park,Calif. (1977), which reference is incorporated herein by reference, andsimilar texts. Examples of polymerases include the large “Klenow”fragment of E. coli DNA polymerase I; Taq polymerase (Cetus)bacteriophage T7 DNA or RNA polymerase, etc. A preferred DNA polymeraseis Taq polymerase (Cetus).

[0061] When an enzymatic reaction, such as a polymerization reaction, isbeing conducted, it is preferable to provide the components required forsuch reaction in “excess” in the reaction vessel. “Excess” in referenceto components of the amplification reaction refers to an amount of eachcomponent such that the ability to achieve the desired amplification isnot substantially limited by the concentration of that component.

[0062] Conditions or agents which increase the rate or the extent ofpriming, primer elongation, or strand displacement, may increase theextent of the amplification obtained with the methods of the presentinvention. For instance, the addition of helicases or single-strandednucleic acid binding proteins may increase the strand displacement rateof a DNA polymerase, or may allow the use of a DNA polymerase that mightnot ordinarily give substantial amplification.

[0063] It is desirable to provide to the assay mixture an amount ofrequired co-factors such as Mg⁺⁺, and dATP, dCTP, dGTP, dTTP, ATP, CTP,GTP, UTP or and the “pre-determined” nucleoside triphosphates insufficient quantity to support the degree of amplification desired.

[0064] Extension of the primer in, for example, a cDNA-containingreaction, may be done with the same reverse transcriptase used to makecDNA. Alternatively, one can add a new DNA polymerase for cDNAextension. Removal of the RNA from the cDNA is preferably done by anRNase H treatment, or by the action of a helicase, but can be done byphysical denaturation, e.g. heat, formamide, or alkali (high pH). In thelatter case, if kinetics of renaturation are sufficiently high, thisstep must be followed by physical separation of the cDNA and RNA or bydegradation of the RNA, e.g. by RNase or alkali. Note that sufficientlyharsh alkali treatment may deaminate dC to form dU, causing a mutation.

[0065] Reverse transcription can be done with a reverse transcriptasethat has RNase H activity. If one uses an enzyme having RNase Hactivity, it may be possible to omit a separate RNase H digestion step,by carefully choosing the reaction conditions.

[0066] All of the enzymes used in this amplification reaction may beactive under the same reaction conditions. Indeed, buffers exist inwhich all enzymes are near their optimal reaction conditions. Therefore,the amplification process of the present invention can be done in asingle reaction volume without, any change of conditions such asaddition of reactants or temperature cycling. Thus, though this processhas several steps at a molecular level, operationally it may have asingle step. Once the reactants are mixed together, one need not addanything or change conditions, e.g. temperature, until the amplificationreaction has exhausted one or more components. During this time, thenucleic acid sequence being amplified will have been increased manyfold. The level of increase will be sufficient for many purposes;however, for some purposes the reaction may have to be repeated withfresh components to achieve the desired level of amplification.

[0067] As discussed above, the degree of amplification obtained throughthe use of PCR is limited if the two primers do not have equivalentefficiency of primer extension. Such a situation is frequentlyencountered, especially in amplification protocols such as RACE,anchored PCR, one-sided PCR, etc. (Frohman, M. A. et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:8998-9002 (1988); Ohara, O. et al., Proc. Natl.Acad. Sci. (U.S.A.) 86:5673-5677 (1989), both of which references areherein incorporated by reference). In brief, these procedures facilitatethe recovery of full-length cDNAs from rare transcripts. The RACEprocedure results in the amplification of sequences 3′ and 5′ of aparticular sequence known to be present in a desired molecule.

[0068] For example in the amplification of mRNA or cDNA molecule havinga 3′ poly-A region, two primers are typically employed. The first primercontains poly-T, and the second primer contains a sequence complementaryto an internal gene sequence of the mRNA or cDNA molecule. Thisprocedure is, referred to as a 3′ RACE (FIG. 1). As shown in FIG. 1A,hybridization with the first primer 3′ poly T 5′ is capable ofhybridizing to the poly-A sequence (FIG. 1B). After primer extension andstrand separation, the structure shown in FIG. 1C is obtained. AfterHybridization with the first primer and with a second primer capable ofhybridizing to a known sequence, the structure shown in FIG. 1D isobtained. Primer extension of both primers yields the structure shown inFIG. 1E.

[0069] Similarly, it is often desirable to amplify a target molecule forwhich only one sequence specific primer is available. This can beaccomplished by adding a nucleotide sequence to one end of the targetmolecule, or complementary copy thereof, and then using a primer whichis complementary to the added nucleotide sequence (FIG. 2). The targetmolecule (FIG. 2A) is hybridized with a first primer capable ofhybridizing to a known sequence (FIG. 2B). After primer extension, andstrand separation or RNAse H degradation of target template thestructure shown in FIG. 2C is obtained. This structure is treated withterminal deoxynucleotidyl transferase and dC to add a poly-dc tail tothe extension product, and the other strand is of no further interest,or has been destroyed by the RNAse H treatment (FIG. 2D). The poly-dCtailed product is amplified by PCR using a poly-dG primer, and a primercapable of hybridizing to the region of known sequence (FIG. 2E). Inpractice, any nucleotide (A, C, T, or G) could have been used to producethe homopolymer tail and be amplified using a complementaryoligonucleotide primer.

[0070] In either of the above examples, the two primers will not havethe same efficiency of primer extension. In the first example, theprimer having a poly-T sequence will have a lower Tm than the secondprimer. In the second example, the poly-dG primer will have a higher Tmthan the other primer. It should be noted that RACE procedures maygenerate artifact products unless nested PCR is done. Nested PCR isdisclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, herein incorporatedby reference. Nested PCR may also be used to eliminate nonspecificamplification products. Note that nested PCR often refers to PCR withprimers “nested” at both ends of the sequence, i.e. PCR conducted using4 oligonucleotides.

[0071] As will be recognized, in both of the preceding examples ahomopolymer primer (i.e. poly-T or poly-dG) is used in conjunction witha second primer having a greater sequence complexity. The presentinvention permits one to make the annealing efficiencies of the twoprimers equivalent by replacing some or all of those natural residues ofthe primers which form 3 hydrogen bonds with pre-determined residuescapable of forming only 2 hydrogen bonds. The number, type and positionof the pre-determined substitute residues is determined (as describedabove) such that the two primers used in the amplification haveequivalent annealing efficiency.

[0072] The primer molecules themselves can be extended by the terminaldeoxynucleotidyl transferase. This is generally an undesired reaction,since it leads to the formation of “primer-dimers” and decreases theefficiency of target molecule amplification. Thus, it is generallypreferable to remove any primer molecules from the reaction prior to thepolynucleotide kinase extension step. Once the step has been completed,the primers may be returned to the reaction. It is thereafterunnecessary to remove the primers after subsequent steps of theamplification.

[0073] The use of such replacement residues in the amplification of apolyadenylated cDNA or mRNA is illustrated in FIG. 3. The figure isidentical to FIG. 1 except for the presence of inosine (represented as a“*”) in the primer and extension product.

[0074] The use of the “pre-determined” nucleotides in the second primerlowers the Tm of that primer, thus permitting it to be equivalent to theTm of the poly-T primer.

[0075] The use of “pre-determined” replacement residues in theamplification of a non-polyadenylated cDNA or mRNA sequence isillustrated below (with the designation “GG*GG*G” referring to a primerhaving at least one “pre-determined” nucleotide such as deoxyinosine)FIG. 4). The figure is identical to FIG. 2 except for the presence ofinosine (represented as a “*”) in the primer and extension product.

[0076] The use of the “pre-determined” nucleotides in the second primerlowers the Tm of that primer, thus permitting it to be equivalent to theTm of the first primer. Note that all of the amplified sequences betweenthe two primer sequences will be the initially present, desiredsequence.

[0077] The present invention is also applicable to amplificationprocedures other than PCR (such as, for example, “Ligation ChainReaction” (“LCR”) described by, for example, Wu, D. Y. et al., Genomics4:560 (1989), or the methods of Miller, H. I. (WO 89/06700), Davey, C.et al. (EP 329,822), Kwoh, D. (Proc. Natl. Acad. Sci. (USA) 86:1173(1989)), etc.). Other suitable methods for amplifying nucleic acid basedon ligation of two oligonucleotides after annealing to complementarynucleic acids are known in the art.

[0078] The method of this invention can be used to adjust (for example,to equalize) the annealing of the oligonucleotides prior to ligation.The ligase based methods have been used for discrimination of targetmolecules which are different by a single nucleotide. The methods of thepresent invention are also applicable for adjusting or equalizing theannealing of oligonucleotides.

[0079] The present invention thus provides a method for adjusting thehybridization efficiency of an oligonucleotide (preferably a primer) ofpre-determined sequence complementary to a target nucleic acid molecule(most preferably a cDNA molecule), which comprises: A) employing as theoligonucleotide an oligonucleotide wherein at least one residue is adeoxyinosine residue, and B) permitting the oligonucleotide to hybridizewith the target molecule. In this method, the presence of thedeoxyinosine residue effects the adjustment of the hybridizationefficiency. As will be appreciated, this adjustment can either increaseor decrease the hybridization efficiency of the respective molecules.

[0080] In the above-described ligation chain reaction (LCR) method, amutation in a target molecule can be detected by using twooligonucleotides each capable of hybridizing to adjacent positions onthe target molecule, such that the positions flank the site of potentialmutation. The sequences of the oligonucleotides is such that if themutation is present, the hybridized molecules will be able to anneal toone another. As will be appreciated, it is readily possible to employoligonucleotide sequences such that the hybridized molecules will beable to anneal to one another if the mutation is not present. Thus, thecapacity of the oligonucleotides to ligate to one another is “probative”of the presence of the mutation.

[0081] The present invention permits one to adjust the relativehybridization efficiency of the two oligonucleotides, by incorporatingdeoxyinosine into one or both oligonucleotides. It is preferable toadjust this relative efficiency to equalize the respective efficienciesof the two oligonucleotides. Such equalization can compensate foroperational constraints caused by differences in the respective G-Ccontent, size, concentration, etc. between the two oligonucleotides.

[0082] In a preferred embodiment, the ligation of the oligonucleotidesresults in the formation of a primer molecule that can be enzymaticallyextended to form a complement of the target molecule. Such amplificationmay be by or include PCR, but will most preferably be mediated by anisothermal extension of the primer to produce the complement to thetarget molecule.

[0083] The present invention may be combined with many other processesin the arts of molecular biology to achieve a specific end. Ofparticular interest is purifying the target sequence from the othersequences in the sample. This can be accomplished most advantageously byannealing the nucleic acid sample to an oligonucleotide that iscomplementary to the target and is immobilized on a solid support. Aconvenient support would be a micro-bead, especially a magneticmicro-bead. After being so bound, the non-target sequences could bewashed away, resulting in a complete or a partial purification.

[0084] After an amplification is performed, one may wish to detect anyamplification products produced. Any number of techniques known to theart may be adapted to this end without undue experimentation.Particularly advantageous in some situations is the capture ofamplification products by an oligonucleotide complementary to a sequencedetermined by the target sequence, the oligonucleotide being bound to asolid support such as a magnetic micro-bead. Preferably, thisoligonucleotide's sequence does not overlap with that of anyoligonucleotide used to purify the target before the amplification.RNA:DNA hybrids formed may then be detected by antibodies that bindRNA:DNA heteroduplexes. Detection of the binding of such antibodies canbe done by a number of methods well known to the art.

[0085] Alternatively, amplified nucleic acid can be detected by gelelectrophoresis, hybridization, or a combination of the two, as is wellunderstood in the art. Those in the art will find that the presentinvention can be adapted to incorporate many detection schemes.

[0086] Sequences amplified according to the methods of the invention maybe purified (for example, by gel electrophoresis, by columnchromatography, by affinity chromatography, by hybridization, etc.) andthe fractions containing the purified products may be subjected tofurther amplification in accordance with the methods of the invention.

[0087] The present invention includes articles of manufacture, such as“kits.” Such kits will, typically, be specially adapted to contain inclose compartmentalization a first container which contains apre-determined nucleotide or a primer containing a pre-determinednucleotide (such as dI); a second container which contains an enzymecapable of adding a nucleotide (capable of base pairing with thepre-determined nucleotide) to a target nucleic acid molecule. The kitmay additionally contain buffers, polymerase or other enzymes,instructional brochures, and the like.

[0088] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE 1

[0089] Methods describing the application of the polymerase chainreaction to the amplification of cDNA-ends derived from low copy numbermRNAs using a single gene specific primer have been described. Reportedas “5′-RACE” (Frohman, M. A. et al., Proc. Natl. Acad. Sci. (U.S.A.)85:8998 (1988)), “anchor PCR” (Loh, E. Y., et al., Science 243:217(1989)), and “one-sided PCR” (Ohara, O. et al., Proc. Matl. Acad. Sci.(U.S.A.) 86:5673 (1989)), these methods, which facilitate the capture ofsequence from 5′-ends of mRNA proceed through the following steps: (1)conversion of specific cDNA using a gene-specific oligonucleotide primer(GSP1); (2) homopolymeric tailing of cDNA with terminal deoxynucleotidyltransferase (TdT); and (3) PCR amplification of tailed cDNA using an“anchor primer” specific for the homopolymer tail, and a second “nested”genespecific oligonucleotide (“GSP2”) which primes upstream of theoriginals primer used for cDNA synthesis. The efficacy ofdeoxyinosine-containing oligonucleotides to prime detailed cDNA in5′-RACE procedures was tested using a model system employing an in vitrotranscribed RNA analyte added to total RNA isolated from HeLa cells. Theenhanced ability of a deoxyinosine-containing anchor primer to carry outspecific PCR amplification of 5′-ends of low copy mRNA from complexmixtures was demonstrated in this system by direct comparison toamplification with an oligo-dG anchor primer.

[0090] Materials and Methods

[0091] General items. SUPERSCRIPT™ RNase H⁻ reverse transcriptase (RT),E. coli RNase H and TdT were from Life Technologies, Inc. Taq DNApolymerase was purchased from Perkin-Elmer Cetus. Buffer components andgeneral reagents were from either GIBCO BRL or Sigma.Deoxyribonucleoside triphosphates and ribonucleoside triphosphates werepurchased as 100 mM solutions from Pharmacia.

[0092] Oligonucleotides. Oligonucleotides were synthesized byphosphoramidite chemistry using an Applied Biosystems model 380Asynthesizer. Oligonucleotides greater than 35 bases were purified bydenaturing polyacrylamide gel electrophoresis and were eluted from thegel matrix essentially as described by Smith, H. O. (Methods Enzymol.65:371 (1980)). other oligonucleotides were used as machine gradepreparations after removal of salts and organics by PD-10chromatography. Molar extinction coefficients and T_(m) calculations foreach oligonucleotide were calculated using the OLIGO computer programfrom National Biosciences (Rychlik, W. and Rhodes, R. E., Nucleic AcidsRes. 17:8543 (1989)). For the GSPI primer, a 24-mer oligonucleotide wasemployed. The GSP2 primer was 21 nucleotides long. Two anchor primers (a“G-Anchor Primer” and a GI-Anchor Primer”) were employed. Both moleculeshad an identical 18 residue long sequence that was complementary to thetarget; and located immediately 5′ to a poly-G sequence of 15 residues(“G-Anchor Primer”) or a poly-GI sequence of 16 residues (“GI-AnchorPrimer”). The poly-G and poly-GI sequences differed in that nucleotides4, 5, 9, 10, 14 and 15 of the poly-GI sequence of the GI-Anchor Primerwere dI, whereas in the G-Anchor Primer, all of the nucleotides of thepoly-G sequence were dG.

[0093] Preparation of RNA Analyte. RNA analyte for 5′-RACE was an invitro transcription product from the gene for chloramphenicol acetyltransferase (CAT) (Horinouchi, S. and Weisblum, B., J. Bacteriol.150:815 (1982)). In vitro transcription was performed using T7 RNApolymerase (GIBCO BRL) according to the manufacturer's recommendations.DNA template was degraded using RNase-free DNase (GIBCO BRL). RNA wasextracted once using a 50:50 mixture of phenol:chloroform and purifiedby Sephadex G-50 (Pharmacia) chromatography. CAT RNA was diluted in DEPCtreated water, and stored at −70° C.

[0094] Preparation of Total HeLa RNA. Total RNA was isolated from HeLacells by guanidinium thiocyanate extraction and equilibriumcentrifugation in CsTFA essentially as described by Okayama, H. et al.(Methods Enzymol. 154:3 (1987)) using an RNA Extraction Kit fromPharmacia.

[0095] 5′-RACE. Varying amounts of in vitro transcribed CAT RNA werecombined with 1 μg of total RNA from HeLa cells, 1 pmole of oligo GSP1,and DEPC treated water in a final volume of 14 μl. Mixtures were heatedat 70° C. for 10 min to denature secondary structure, and then chilledon ice. Following a brief centrifugation, the remainder of the firststrand synthesis components was added. Reactions were equilibrated to42° C. for 2 min prior to the addition of SUPERSCRIPT RT. First strandsyntheses were performed in final volumes of 20 μl consisting of 20 mMTris—HCl (pH 8.4), 50 mm KCl, 2.5 mM MgCl₂, 100 μg/ml BSA, 10 mM DTT, 1pmole GSP1, RNA and 200 units SUPERSCRIPT RT and were incubated 30 minat 42° C. Following first strand conversion, reactions were equilibratedto 55° C. and 2 units E. coli RNase H was added to destroy CAT RNAtemplate. Specific cDNA products were purified, tailed with dCTP, andamplified by PCR as described below.

[0096] Primer and unincorporated dNTPs were separated from cDNA using a30K low-binding Ultrafee-MC filter unit from Millipore essentiallyaccording to the manufacturer's recommendations. Four successive washsteps with 350 μl 0.1X TE buffer were used to insure sufficient removalof first stand primer. Centrifugations were at 2,000×g for 5 min.

[0097] Purified cDNA was recovered with a final rinse of 50 μl sterileH₂O, transferred to a 0.5 ml microtube, lyophilized till dry using aSavant Speed-Vac, then dissolved in 19 μl 10 mM Tris—HCl (pH 8.4), 25 mMKCl, 1.25 mM MgCl₂, 50 μg/ml BSA, and 200 μM dCTP. The mixture wasdenatured 2 min, 94° C., then chilled on ice, and the contents collectedby brief centrifugation. Homopolymeric tailing was initiated by additionof 10 units TdT and incubated 5 min, 37° C., then 10 min, 65° C.

[0098] Tailed cDNA was amplified directly from TdT reactions withoutprior dilution. Following a brief centrifugation to collect tailed cDNA,one-tenth of each tailing reaction, 2 μl aliquots, from were amplifiedby PCR. Amplification reactions were performed in 50 μl volumes composedof 20 mM Tris—HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA], 0.2mM each dNTP, 4 pmoles GSP2, 4 pmoles Anchor Primer (either theGI-Anchor Primer or the G-Anchor Primer), tailed cDNA and 1 unit Taq DNApolymerase (Perkin Elmer-Cetus). Reactions were assembled on ice,overlaid with 50 μl light mineral oil (Sigma), and placed into a DNAThermalcycler (Perkin Elmer Cetus) which had been equilibrated to 94° C.Following an initial 5 min denaturation at 94° C., PCRs were temperaturecycled through 30 cycles as follows: 45 s at 94° C. (denaturation); 25 sat 55° C. (annealing); and 3 min at 72° C. (extension). After the finalcycle an additional 7 min. extension at 72° C. was performed and thenreactions were held at 4° C.

[0099] Control reactions omitted either RNA analyte, reversetranscriptase, or TdT. A quantified detailed cDNA target, derived fromthe CAT RNA analyte, was used as a positive control.

[0100] Analysis of Amplification Products. Following amplificationone-fifth, 10 μl of each PCR was analyzed by electrophoresis on a 1.5%agarose/TBE gel. Amplified DNA was visualized by ethidium bromidestaining and photographed.

[0101] Results

[0102] Based on sequence analysis of the CAT RNA analyte, primers usedfor amplification were predicted to produce a 930 bp product fromdC-tailed CAT cDNA target. This product was observed in PCRs primed withGSP2 and either the GI-anchor primer or the G-anchor primer. However,sensitivity and band intensity was approximately 10 fold greater in PCRscontaining equivalent amounts of target which were amplified using theGI-anchor primer as compared to amplifications which used the G-anchorprimer. The 930 bp product was clearly visible in 5′-RACE reactionsinitiated with as few as 10⁵ copies of RNA analyte when the GI-anchorprimer was used for amplification. However, product was not visible in aparallel reaction amplified with the G-anchor primer. Band intensity ofthe 930 bp product resulting from 5′-RACE reactions containing 10⁶copies of CAT RNA analyte was approximately 10 fold greater in PCRsperformed with the GI-anchor primer as compared to PCRs primed with theG-anchor primer.

[0103] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A method for amplifying the concentration of anucleic acid molecule using two primers, comprising the steps: (a)performing the template-dependent extension of a first primer, saidprimer being hybridized to a first strand of said molecule, wherein saidextension forms a second strand of a nucleic acid molecule complementaryto said first strand; (b) performing the template-dependent extension ofsaid second strand, by extending a second primer, said primer beinghybridized to said second strand of said molecule, wherein saidextension forms a copy of said first strand of said nucleic acidmolecule; (c) performing the template-dependent extension of said copyof said first strand, to thereby form a copy of said second strand ofsaid nucleic acid molecule; (d) repeating steps (a), (b), and (c), tothereby achieve said amplification of said nucleic acid molecule;wherein at least one of said first and second primers contains; at leastone deoxyinosine residue, and wherein said first and second primers haveequivalent efficiency of primer extension.
 2. The method of claim 1,wherein said nucleic acid molecule is an RNA molecule.
 3. The method ofclaim 2, wherein said RNA molecule is single-stranded.
 4. The method ofclaim 2, wherein said RNA molecule is double-stranded.
 5. The method ofclaim 1, wherein said nucleic acid molecule is a DNA molecule.
 6. Themethod of claim 5, wherein said DNA molecule is single-stranded.
 7. Themethod of claim 5, wherein said DNA molecule is double-stranded.
 8. Themethod of claim 1, wherein only one of said primers contains at leastone deoxyinosine residue.
 9. The method of claim 1, wherein both of saidprimers contain at least one deoxyinosine residue.
 10. The method ofclaim 1, wherein said nucleic acid molecule being amplified ispolyadenylated at its 3′ end, and wherein one of said primers contains apoly-T sequence, and the other of said primers contains at least onedeoxyinosine residue.
 11. The method of claim 1, wherein said nucleicacid molecule being amplified, copy thereof or complementary copythereof, has been extended to contain a 3′ sequence, and wherein one ofsaid primers is capable of hybridizing to said 3′ sequence, said primercontaining at least one deoxyinosine residue.
 12. The method of claim 1,wherein at least one of said primers is extended using a thermostableDNA polymerase.
 13. The method of claim 12, wherein said thermostableDNA polymerase is Taq polymerase.
 14. A kit for amplifying a nucleicacid molecule containing: a first container containing a primer, saidprimer being capable of hybridizing to said nucleic acid molecule, andcontaining at least one deoxyinosine residue; and a second containercontaining an enzyme capable of adding a C nucleotide to said nucleicacid molecule, said C nucleotide being capable of base pairing with saiddeoxyinosine residue of said primer.
 15. The kit of claim 14, whichadditionally contains a third container containing a thermostable DNApolymerase capable of extending the primer of said first container, whensaid primer is hybridized to a sequence containing said C residue addedby the enzyme of said second container.
 16. The kit of claim 15, whereinsaid thermostable DNA polymerase is Taq polymerase.
 17. A kit foramplifying a nucleic acid molecule containing: a first containercontaining a first primer, said primer being capable of hybridizing tosaid nucleic acid molecule, and containing at least one deoxyinosineresidue; and a second container containing a second primer; whereintemplate-dependent extension of said first primer produces a secondnucleic acid molecule which is capable of hybridizing to said secondprimer, and wherein template-dependent extension of said second primerproduces a copy of said first nucleic acid molecule.
 18. The kit ofclaim 17, which additionally contains a third container containing athermostable DNA polymerase capable of extending either the primer ofsaid first container, or the primer of said second container when saidprimer is hybridized to a nucleic acid molecule.
 19. The kit of claim18, wherein said thermostable DNA polymerase is Taq polymerase.
 20. Amethod for adjusting the hybridization efficiency of an oligonucleotidehaving a pre-determined sequence complementary to a target nucleic acidmolecule, which comprises: A) employing as said oligonucleotide anoligonucleotide wherein at least one residue is a deoxyinosine residue;B) permitting said oligonucleotide to hybridize with said targetmolecule, wherein the presence of said deoxyinosine residue effects saidadjustment of said hybridization efficiency.
 21. The method of claim 20,wherein said target nucleic acid is a cDNA molecule.
 22. The method ofclaim 21, wherein a first and a second oligonucleotide are employed,said first oligonucleotide being said oligonucleotide having at leastone deoxyinosine residue, and wherein said oligonucleotides are capableof hybridizing to said target molecules at adjacent positions, such thatthey may be ligated to one another.
 23. The method of claim 20, whereinsaid oligonucleotides when ligated together form a primer, and whereinsaid primer is isothermally extended to produce a complement to saidtarget molecule.
 24. The method of claim 22 wherein the capacity of saidoligonucleotides to ligate to one another is probative of the presenceof a mutation.